Managing mortality: how cells regulate telomerase activity

Telomere length is a key determinant of the normal lifespan of a cell, and dysfunction in telomere maintenance has been linked to several disorders as well as cancer via genomic instability. Therefore, the mechanisms governing telomere elongation by telomerase have been a topic of considerable interest in recent years. Several new studies shed new light on how telomerase is recruited and controlled, and one implicates the CST complex, comprising CTC1, STN1, and TEN1 in mammalian cells, in shutting off telomerase to restrict telomeric elongation. These studies continue to enhance our understanding of how telomere maintenance is regulated, an understanding that may guide development of future therapies for cancer and other telomere-related diseases.

Highlights

Telomerase activity is crucial in telomere maintenance in proliferative cells such as stem and cancer cells

Telomeres are repeats of a short DNA sequence, positioned at the ends of linear chromosomes, that protect coding DNA from being lost during successive rounds of cell division due to the end replication problem. With each division of a cell, its telomeres shorten, and therefore the length of a cell's telomeres gives information as to its proliferative age. When telomere length reaches a certain limit, the cell will either die or undergo senescence (1–3).

Several protein complexes are involved in the protection and maintenance of telomeres. Shelterin is a complex of 6 proteins in vertebrates, TRF1 and 2, TIN2, Rap1, TPP1, and POT1, that binds telomeres and plays several roles, including assistance in formation of the t-loop structure and telomere end protection, as well as telomerase recruitment (see figure The CST complex limits elongation by telomerase) (3–5). Telomerase, a ribonucleoprotein (RNP) expressed in a limited number of cell types including stem cells and cancer cells, replenishes telomeres at their 3' single-strand ends. Telomerase comprises the TERT component, a reverse transcriptase, and the TERC component, an RNA template for the telomeric repeat sequence, as well as additional proteins like dyskerin, which stabilizes the complex (3).

Because telomere function is so crucial to cellular lifespan, dysfunction has serious implications for a range of diseases. Short or absent telomeres, which can lead to chromosomal instability via end-to-end fusions and other events when DNA checkpoints are defective, are associated with many different types of cancer. Telomerase upregulation is also noted in many cancer cells, suggesting that this is the mechanism by which these cells acquire proliferative immortality (2–3). Mutations in the key genes involved in telomere maintenance are also linked to serious consequences. Dyskeratosis congenita, which causes bone marrow failure, is the result of mutations in one of two components of the telomerase complex, DKC1 or TERC. Other telomerase mutations lead to adult-onset aplastic anemia, pulmonary fibrosis, or liver cirrhosis (2). Research into the mechanisms that govern telomere maintenance, therefore, is potentially important for developing therapies for a variety of diseases.

Regulatory mechanisms influencing telomerase activity continue to be elucidated. Very recently, the multi-functional energy enzyme GAPDH was shown to drive senescence and telomere shortening in cancer cells. The Rossman fold in the GAPDH N-terminal region mediated binding with TERC, the telomerase RNA component, and its C-terminal region inhibited telomerase activity (6). Another recent study demonstrated that the OB-fold domain of TPP1, a shelterin protein, is intimately involved in telomerase recruitment, and identified specific residues responsible for this activity (5). The study additionally identified specific mutations in TERT that prevent recruitment via this interaction and are also associated with idiopathic pulmonary fibrosis.

Back to top CST regulates telomerase activity and telomere elongation in human cells

While several studies have examined the regulation of telomerase, how exactly telomerase activity shuts off to prevent over-elongation had been unclear. A recent study implicates the human CST complex in this process (7). The CST complex has been recognized for some time as a key player in telomere maintenance; previous studies had shown that CST bound the G tail in budding yeast, mediating telomere protection, regulation of the length of the G-tail overhang possibly via facilitation of C-strand synthesis, and telomerase recruitment (8). In recent years, a CST function in telomere protection had been explored in other organisms, including plants and mammals (8-10). More recently, CST was shown to play a role in fill-in synthesis following 5' end resection by the nuclease Exo1, correcting the length of the 3' overhang after its lengthening in the S phase of the cell cycle (11).

Chen et al. set out to more fully characterize how CST affected telomere length, first using shRNA knockdown in human telomerase-expressing fibrosarcoma cells to show the importance of each of the CST components for restricting telomere elongation. Retrovirally transducing CST fragments into cells with or without telomerase, they observed that the amino-terminal CST fragment only shortened telomeres in telomerase-positive cells — therefore, its impact on length was due to interaction with telomerase. The carboxy-terminal fragment induced telomere lengthening in these cells, but this was blocked when telomerase was inhibited, further strengthening the connection between CST, telomerase, and telomere length regulation. EMSA experiments demonstrated that the CST complex bound readily to G-strand DNA 18-mer and longer.

The next step was to determine the nature of CST's ability to block telomerase function. In vitro, direct telomerase activity experiments showed that CST needed to bind primer substrate to block telomerase activity. However, in the presence of POT1-TPP1, which stimulates telomerase, this requirement was unnecessary, suggesting a second mechanism of telomerase inhibition by CST. To ensure that the diminished amount of telomerase activity was not simply due to CST blocking the binding of free POT1-TPP1 to new product, the team bound POT1-TPP1 to primer and washed away excess protein, then observed whether or not CST could still inhibit telomerase activity when POT1-TPP1 was already bound. Indeed, CST continued to block new synthesis, and yeast two-hybrid experiments revealed physical interactions between CST components and POT1-TPP1. Therefore, CST can block telomerase by either directly binding to substrate, or by interacting with POT1-TPP1.

To confirm these associations in the cell, the team used ChIP in cancer cells to show that CTC1, one of the three proteins comprising CST, associates with telomeric DNA throughout the cell cycle, and competes with POT1 and TPP1 for binding. Telomerase activity ceases in late S and early G2 phase, and indeed, this is when CTC1 association with telomeric DNA is highest. Telomerase inhibition blocked the increase in CTC1 binding, suggesting that telomerase activity and the 3' overhang it generates is a requirement for CST binding. Overall, the team concluded that CST is a shutoff mechanism for telomerase elongation, keeping the process in check during each cell cycle to prevent overelongation.

The ability of telomerase to replenish telomeric repeats in proliferative cells is crucial for these cells's function, and also contributes to the immortality of cancer cells. Continuing research to define the mechanisms of telomere maintenance and elongation has yielded insights into such important aspects of the process as telomerase recruitment and cessation of telomerase activity, but many questions still remain. Further characterizing these processes will be useful in developing treatments for cancer and diseases resulting from defective telomere-associated proteins.